Brain function relies on the coordinated actions of trillions of tiny specialized structures called synapses, which serve as communication gateways between neurons. Small changes in these synaptic machineries are the early manifestation of many neurological disorders and neurodegenerative diseases, which become increasingly prevalent as the population ages. Proper synaptic function relies on the precise, yet dynamic, regulation of the abundance and subcellular localization of synaptic proteins. Monitoring the spatiotemporal organization of these proteins in live neurons under native conditions is an important step toward understanding their function. However, it remains challenging to visualize endogenous synaptic protein organization in neurons in their native habitat - in living brain tissue or in living animals. The majority of studies investigating protein dynamics rely on the overexpression of fluorescently tagged proteins of interest. Unfortunately, overexpression can alter protein stoichiometry, trafficking, subcellular localization, and signaling, ultimately affecing cellular function. Although 'knock-in' strategies can, in principle, be used to label the target molecule with a fluorescent protein and express it at endogenous levels, most knock-in approaches result in the global expression of the labeled protein. Global expression leads to high background fluorescence and a lack of cell-specific contrast in intact tissue, making high-resolution imaging studies of protein dynamics difficult. To address these problems we propose to develop an innovative mouse knock-in strategy called Conditional Labeling by Exon Duplication, or CLED, to fluorescently tag specific proteins and express them at endogenous levels in a sparse subset of neurons. By using an innovative approach that combines the Cre/LoxP site- specific recombination strategy with exon duplication, we will express the tagged proteins under the control of endogenous transcriptional and translational regulation while maintaining cell-specific, Golgi-staining-like high contrast. As a proof of principle, in Specific im 1, we will tag the critical synaptic protein PSD-95 with the yellow fluorescent protein mVenus, and, in Specific Aim 2, we will use the resulting transgenic mouse line to examine functionally significant dynamics of PSD-95 organization. Once established, our proposed strategy will complement current microscopy techniques and sample preparation methods to provide a previously unattainable, highly sensitive, dynamic view of the spatiotemporal organization of specific synaptic proteins in situ and in vivo. The same strategy can be applied to visualize proteins that carry out non-neuronal functions. The acquired knowledge and principles from these novel transgenic mouse lines will facilitate future studies of human brain function and disease.